Detection Of Liposoluble Algal Toxins In Offshore Sediments And Suspended Particles And Purification Of Toxins In Algae

During the past decade, lipophilic algae toxins（LATs） in the marine environment have become a hot research topic due to their wide distribution and great influence on the marine ecological environment. However, few studies on the presence of LATs in sediments and suspended particles collected from seawater were conducted, and an effective method for determining LATs in sediments and suspended particles was unavailable. Therefore, it is necessary to establish a rapid and highly efficient method for the determination of LATs in sediments and suspended particles to clarify the existing forms and environmental behavior of these toxins in the marine environment. LATs standards with high purity were of vital necessity in the detection of chemical method and it also restricted the studies of environmental chemical behavior, toxic mechanism, and other related fields. Therefore, purification technology of LATs urgently needs to be solved by scientific researchers. In this study, high performance liquid chromatography-mass spectrometry（HPLC-MS） for the determination and quantification of LATs in sediments and suspended particles was developed, and real samples were detected. Besides that, using prorocentrum lima cultured in the laboratory as raw material, feasibility for the purification of the three typical LATs by high speed countercurrent chromatography-tandem mass spectrometry（HSCCC-MS） were conducted. The details are as follows:1. A method based on sample preparation by accelerated solvent extraction（ASE） and analysis by high performance liquid chromatography-tandem mass spectrometry was developed and used for the determination of seven typical LATs in marine sediment samples collected from three coastal bays in China. The sediment samples were pretreated by ASE and a series of technical indexes were optimized. The qualitative and quantitative analysis of seven LATs（Okadaic acid（OA）, Dinophysistoxins-1（DTX1）, Spirolide-1（SPX1）, Yessotoxin（YTX）, Azaspiracid-1（AZA1）, Azaspiracid-2（AZA2）, and Pectenotoxin-2（PTX2）） were performed on HPLC-ESI-MS₂ in multiple reaction monitoring（MRM） mode. In addition, matrix-matched calibration curves were prepared and used during quantification to eliminate matrix effects. Experimental results show that ASE as an extraction technology offers a satisfactory extraction result for seven LATs in sediment. Under the optimal conditions, satisfactory specificity, reproducibility（relative standard deviations（RSDs） ≤ 14.76%）, stability（RSDs ≤ 17.37%）, recovery（78.0%–109.0%）, and detection limit（34.4 pg/g–618.5 pg/g） of the method were achieved. These results suggested that the proposed method with good sensitivity and accuracy was reliable for simultaneously determining the seven LATs in marine sediments. Different concentrations of OA and PTX2 were detected from Hangzhou bay, Jiaozhou bay and Laizhou bay by the proposed method. Samples from Hangzhou bay revealed OA as the predominant LATs with concentrations ranging from 186.0 to 280.7 pg/g; PTX2 was the main LATs in sediment samples from Laizhou bay at the concentrations from 256.4 to 944.9 pg/g; OA and PTX2 were detected from Jiaozhou bay and the concentrations were both below the limit of quantitation. These results suggested that the sediments obtained from Hangzhou bay, Laizhou bay and Jiaozhou bay were all contaminated by certain amounts of LATs.2. A method was developed for simultaneous determination of the eight typical LATs in suspended particles by ultra performance liquid chromatography-electrospray ionization-mass spectrometry（UPLC-ESI-MS）. After being extracted with methanol by ultrasonic-assisted extraction,the extracts were performed on an Acquity UPLC BEH C18 column（2.1 mm×50 mm,1.7 mm） using gradient elution of acetonitrile and water containing 5 mmoL/L ammonium acetate as eluent modifiers. Qualitative and quantitative analysis of samples were carried out by electrospray ionization-mass spectrometry（ESI-MS₂） in MRM mode. Under the optimal conditions, eight LATs（OA, Gymnodimine（GYM）, DTX1, SPX1, YTX, AZA1, AZA2, and PTX2） were well-separated within 5 min. Satisfactory precision（RSDs≤ 14.13%）, the recoveries（83.8- 98.3%）, and detection limits（2.9- 103.1 pg/g） of the method were achieved. Good linearity（at least R2 ≥0.99） was also obtained for all studied analytes. These results suggested that the proposed method was reliable for simultaneously determining the eight LATs in suspended particle collected from seawater. Then, the method was applied to determine the amounts of the eight LATs in authentic suspended particle samples collected from Qingdao near-shore area. PTX2 was detected in the samples from Shilaoren beach and No. 3 bathing beach at concentrations of 717.2 and 790.6 pg/g（dry weight）, respectively.3. A method was developed for separation and purification of three LATs from prorocentrum lima by HSCCC-MS, combined with macroporous resin and semi-preparative HPLC. The three LATs in prorocentrum lima were extracted with 90% methanol aqueous solution by ultrasonic-assisted extraction and then preliminary purified with macroporous resin. After optimized a series of technical indexes of HSCCC, HSCCC-MS was adopted to separate the three LATs in crude extraction handle with macroporous resin. Lastly, further purification was performed by semi-preparative HPLC for the three LATs. Experimental results show that the solvent system of n-hexane/ ethyl acetate/ methanol/ 1% formic acid aqueous（1∶1.75∶1.2∶1,V/V） was optimization for separating the three LATs by HSCCC,and this method shows good repeatablity and stability with sample load in 150 mg at a time. Compounds isolated from crude extraction by HSCCC showed excellent purity. The purity of OA, compound 816 and DTX1 was 89%, 86% and 83%, respectively. Further purification by semi-preparative HPLC increased the purity of the three LATs above 99%. The structures of purified toxins are easy to alienate in acidic solutions and the compounds were stable in dry powder under low temperature.